Re-engineering the two-component systems as light-regulated in Escherichia coli

Area of Science:

• Microbiology
• Molecular Biology
• Systems Biology

Background:

• Bacteria utilize two-component systems (TCSs) for sensing and responding to environmental changes.
• TCSs consist of histidine protein kinases (HKs) and response regulator proteins (RRs) coupled by phosphorylation.
• Understanding TCS function is crucial for comprehending bacterial adaptation.

Purpose of the Study:

• To develop an optogenetic approach for controlling bacterial TCSs.
• To create a library of light-responsive HK-Cph1 chimeras for 16 different TCSs.
• To demonstrate the light responsiveness and frequency response of engineered TCSs.

Main Methods:

• Re-engineering sensor histidine protein kinases (HKs) in *Escherichia coli* using an optogenetics approach.
• Constructing light-sensing fusion proteins by replacing the native sensing domain of HKs with the Cph1 light-sensing domain from *Cyanobacteria Synechocystis*.
• Creating a library of 16 HK-Cph1 chimeras and testing the NarX-NarL system's light responsiveness and frequency response.

Main Results:

• Successfully constructed a library of light-controllable HK-Cph1 chimeras for 16 HK-RR TCSs.
• Demonstrated red light responsiveness of the engineered NarX-NarL system.
• Investigated the frequency response characteristics of the light-controlled NarX-NarL TCS.

Conclusions:

• The developed library provides a versatile toolkit for studying TCSs using optogenetics.
• This approach enables precise, light-based control over bacterial signaling pathways.
• The findings pave the way for novel investigations into bacterial physiology and adaptation mechanisms.